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Perit Dial Int 27(Supplement_2): 72-75
2007
© 2007 International Society for Peritoneal Dialysis
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Inflammation and Fibrosis

IN VITRO STUDY OF PERITONEAL FIBROSIS

Kuan-Yu Hung, Kuan-Dun Wu and Tun-Jun Tsai

Department of Internal Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan

Correspondence to: T.J. Tsai, Department of Internal Medicine, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei, Taiwan. paul{at}ha.mc.ntu.edu.tw


   ABSTRACT
TOP
 ABSTRACT
DISCUSSION
 CONCLUSIONS
 REFERENCES
 

Peritoneal fibrosis (PF) is an important issue in peritoneal dialysis (PD) because it remains one of the leading causes of patient drop-out from PD. In this review, we focus on in vitro approaches to the pathogenesis and therapeutic potential of PF and on associated clinical implications. Representative Asian studies, initiated since mid-1990s, that have investigated matrix accumulation in peritoneal tissue possibly leading to PF in the PD population will be highlighted as examples to learn how to apply this research tool. As compared with data from well-designed clinical trials, observations from in vitro models may be far from becoming solid evidence; however, they do cast new light on options for investigations into therapeutic pharmaceuticals.

KEY WORDS: In vitro study; peritoneal fibrosis; mesothelial cell; fibroblast; cytokine; tamoxifen.

Why is peritoneal fibrosis an important issue in renal care?

Peritoneal dialysis (PD) has been well-recognized as a major mode of renal replacement therapy for almost 20 years (1). However, a large proportion of patients still drop out from PD because of technique failure. In Taiwan, based on two years of observation of a small PD cohort, we found that about 16% of patients experience technique failure (2).

Except for refractory or severely complicated peritonitis, ultrafiltration failure is the leading cause of dropout from PD (3). Ultrafiltration failure most likely results from alterations in peritoneal anatomy and function secondary to exposure to glucose or to bioincompatible PD fluids (4).

The term "peritoneal fibrosing syndrome" (PFS) represents a wide range of peritoneal structural changes—resembling peritoneal fibrosis—observed in long-term PD patients (5). An extreme example of PFS is encapsulating peritoneal sclerosis, which is associated with poor outcome and high mortality (6–8). In the present article, we focus on in vitro approaches to the pathogenesis and therapeutic potential of peritoneal fibrosis and on associated clinical implications.


   DISCUSSION
TOP
 ABSTRACT
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
PATHOGENESIS OF PERITONEAL FIBROSIS
The human peritoneal cavity is covered by a thin (30 – 40 µm) layer of normal peritoneum, composed of a superficial monolayer of peritoneal mesothelial cells (PMCs) and the submesothelial tissue (9). With time on PD, degenerative changes in the PMCs, matrix accumulation in the submesothelial tissue, and vasculopathy in peritoneal tissue become dominant. The factors believed to mediate fibrogenic reaction in peritoneum are the high glucose content, hyperosmolarity, and low pH of PD solutions and the glucose degradation products (GDPs) and advanced glycosylation end products (AGEs) encountered during the process of PD therapy (10).

Figure 1 shows the multiple sources of cytokines and growth factors postulated to mediate the development of peritoneal fibrosis in long-term PD patients. The main and common final presentation of peritoneal fibrosis is an inappropriate accumulation of matrix within peritoneal tissue. Here we discuss representative studies that have used in vitro models to explore the possible mechanisms and therapeutic potential of peritoneal fibrosis. For a fuller discussion of experimental techniques, readers can consult two in-depth reviews recently published in Peritoneal Dialysis International (11,12). Table 1 outlines major aspects of in vitro models of study that apply to investigations of peritoneal fibrosis.


Figure 1
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  		Figure 1 — The cytokines and growth factors that chiefly mediate the development of peritoneal fibrosis in peritoneal dialysis (PD) patients. GDPs = glucose degradation products; AGEs = advanced glycosylation end-products; IL = interleukin; TNF = tumor necrosis factor; TGF = transforming growth factor; CTGF = connective tissue growth factor; VEGF = vascular endothelial growth factor.

 

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  		TABLE 1 Major Aspects of Studies into Peritoneal Fibrosis to Which In Vitro Models Can Be Applied

 

IN VITRO STUDIES OF PERITONEAL FIBROSIS: ASIAN EXPERIENCE
Representative in vitro studies from Asia that have investigated matrix accumulation in peritoneal tissue possibly leading to peritoneal fibrosis in the PD population show how this tool can be applied to PD research. For simplicity, the pathophysiologic process described in Figure 1 has been translated into disquisitive categories (Figure 2).


Figure 2
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  		Figure 2 — Main categories of in vitro studies applied to explore possible mechanisms and therapeutic potentials of peritoneal fibrosis (PF).

 
Who Executes the Process?: Peritoneal fibroblasts and PMCs are the main guardians in the peritoneal cavity (11,12). They participate actively and take charge of peritoneal defense during the process of PD. Investigating the roles of PMCs and fibroblasts in the development of peritoneal fibrosis is therefore a reasonable approach.

Since in mid-1990s, along with other Asian investigators (13–16), we (17,18) became interested in the new experimental approach of the cell-culture model. Asian experiences began to accrue showing that PMCs (13,14, 17,18) and peritoneal fibroblasts (13) are both involved in matrix synthesis (16,19) when exposed to PD effluent, high glucose stimuli (15), AGEs (20), and GDPs (21). Epithelial-to-mesenchymal transition (EMT) in PMCs is an important issue linking these two cell populations (22,23), but this relatively new field of investigation remains less explored in Asia (21,24).

Cytokines and Growth Factors: Who Are They?: The downstream factors and signaling transduction pathways that mediate the development of peritoneal fibrosis are worthy of study both for elucidating mechanisms and for discovering useful drugs. Pivotal clinical studies by Krediet and colleagues have demonstrated that vascular endothelial growth factor (VEGF) and transforming growth factor beta (TGF-β) are major mediators in the development of peritoneal fibrosis (25,26).

To further explore the molecular mechanisms and signaling pathways in peritoneal fibrosis, in vitro models were adopted. Those models revealed that protein kinase C (27,28), mitogen-activated protein kinases (29–32), the SMAD pathway (30,31), and the downstream connective tissue growth factor (CTGF) are important in matrix synthesis (33,34). Agents that increase intracellular cyclic adenosine monophosphate may have a suppressive effect on TGF-β and subsequent peritoneal fibrosis processes (18,29–31). Recently, Lee and colleagues from Korea pioneered in vitro cell-culture studies in Asia on the roles of reactive oxygen species (28) and angiotensin II (35) in high-glucose–induced matrix synthesis by PMCs and on the resulting impact on the pathogenesis of peritoneal fibrosis. Our own preliminary in vitro experiments observed that hypoxia-inducing factor can modulate expression of VEGF, TGF-β, and CTGF in high-glucose–treated or angiotensin II–stimulated PMCs (unpublished data). More in vitro and in vivo studies are expected to further elucidate the complex networks operating at the cellular level.

Can We Reduce or Reverse Matrix Accumulation?:Matrix accumulation is a final result of the counterbalance between synthesis (production) and degradation (proteolysis or fibrolysis) of matrix components in peritoneal tissue. Agents such as dipyridamole (29,30), pentoxifylline (31), steroids (36), emodin (37,38), and pyridoxamine (39) have all been tested in cell-culture models of PMCs or peritoneal fibroblasts, achieving varying degrees of suppressive effects on matrix synthesis. As compared with data from well-designed clinical trials, observations from the foregoing in vitro models may be far from becoming solid evidence; however, they do cast new light on options for investigations into therapeutic pharmaceuticals.

Is Everything Fine? (The Safety Issues): Another useful, but commonly forgotten, application of in vitro models is testing for drug safety. Take tamoxifen as an example. Reports indicated that tamoxifen might be useful in the management of peritoneal fibrosis (40–43); however, in our limited observations in an high-glucose–treated PMC culture model, we noticed accelerated apoptosis of PMCs in the presence of tamoxifen (unpublished data). Our group will probably hold tamoxifen aside, waiting until safety issues been better clarified, before we recommend its use intraperitoneally in patients. On the other hand, our group has recently completed in vivo experiments examining the efficiency and safety of tamoxifen in a rat model of PD. In vitro models, though relatively less conclusive, provoke more questions than answers and typically illuminate options worth further research.


   CONCLUSIONS
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 ABSTRACT
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
We hope to have convinced readers, through this brief but integrated review on the application of in vitro studies for peritoneal fibrosis, that these models still stand as a powerful and convenient point from which to predict the way forward (in vivo models, therapeutic trials). A Chinese proverb says "To go far, one must start from near." But this advice holds good only when questions are asked and appropriate methods are applied.


   ACKNOWLEDGMENTS
 
This review was supported by grants from the National Science Council (NSC 94-2314-B-002-318) and the Mrs. Hsiu-Chin Lee Kidney Research Fund. The authors would like to thank Ms. Shin-Yun Liu and Dr. Jenq-Wen Huang for their contributions in manuscript preparation and technical assistance.


   REFERENCES
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 ABSTRACT
 DISCUSSION
 CONCLUSIONS
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K.-Y. Hung, J.-W. Huang, C.-K. Chiang, and T.-J. Tsai
Preservation of peritoneal morphology and function by pentoxifylline in a rat model of peritoneal dialysis: molecular studies
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